It is generally accepted that tolerance to organ-specific self-antigens develops in a programmed fashion early in ontogeny by active suppression and/or deletion of potentially self-reactive clones of lymphocytes. Although few studies documenting the timing of these events in vivo have been published, a now classic series of experiments by Edward Triplett (1962) does bear directly on this issue. Triplett removed the anlage of the pituitary from tailbud tree frog embryos. He "parked" these anlagen on intermediate hosts until the immune system of the original hypophysectomized host had developed and then transplanted the differentiated gland back to its original host. That such grafts were rejected by hosts whose immune systems had matured in the absence of the pituitary, has been taken as strong evidence for the early development of self tolerance to organ-specific antigens. Our preliminary studies with frog embryos and this model system, have produced significantly different results. Removal of eye and pituitary anlagen, followed by grafting of self eyes or pituitaries to organ-deprived frogs during larval and post-metamorphic life, have not resulted in self rejection. We propose to use this unique amphibian model system to re-examine the question of when self-tolerance to organ-specific antigens is acquired. We will study the eye, pituitary, and thyroid as test organs, but thyroid will be studied in more depth than eye or pituitary. The anlagen of these organs will be extirpated early in embryonic life, before any development of the immune system has occurred. The anlagen will be discarded and replaced at a later time by differentiated organs from isogenic frogs. If unresponsiveness is observed at a given stage, we will conduct experiments to determine whether it is due to tolerance induction or simply due to failure of the immune system to contact the relevant organ-specific antigens. If exposure of the immune system has been confirmed, yet unresponsiveness is still observed, we will conclude that the exposure has led to tolerance induction. In an effort to probe the mechanisms underlying self-tolerance, we will attempt to interfere with development of tolerance and/or try to "break" tolerance by transfer of specifically reactive lymphocytes. If rejection of self by an organ-deprived host occurs we shall use such animals as sources of anti-self reagents for adoptive immunization protocols in an attempt to better understand self tolerance in normal intact animals.